1. Field of the Invention
The present invention generally relates to the technical field of molecular biology. More specifically, the invention relates to method for detecting and identifying peptides involved in protein-peptide interaction in cellular systems. In particular, the present invention relates to methods for detecting, identifying and optionally isolating peptides such as functional peptide ligands of target proteins.
2. Description of Related Art
Peptides show a wide variety of biological activities in cells one important example of which are natural peptide hormones acting as important molecular signals by binding to cellular receptors. It has also been shown that synthetic peptides can modulate the activity of proteins and even inhibit protein-protein interactions and are therefore important lead compounds in the field of drug discovery. Due to their high activity and specificity, during the last years there was an increased focus on peptides as therapeutic effectors.
The development of methods for detecting interactions between target proteins and peptides under physiological conditions is of major interest. Although there exists already a number of methods for detecting interactions between proteins and peptide ligands as well as for isolating novel peptide ligands, in particular, methods for identifying novel peptide ligands are of special interest in drug discovery. However, existing in vitro methods for the identification of binding peptides (Liu et al., Exp. Hematology 31 (2003), 11-30) require as a prerequisite the purification of the protein of interest, resulting in most cases in a decreased biological activity and binding quality, since many target proteins adopt their preferred conformation exclusively in the cytoplasm which can differ from their conformation under non-physiological conditions.
One common method for isolating peptide ligands allowing the presentation of peptide libraries on the surface of a filamentous phage (Smith, Science 228 (1985), 1315-1317), comprises the introduction of exogenous peptide sequences into the genome of phage capsid proteins, because of which this method is also referred to as phage display technology. International application WO95/31723 describes the use of this method for in vitro selection of peptides from biological peptide libraries, but although there are few examples for its application to living organisms (Arap et al., Proc. Natl. Am. Soc. 99 (2002), 1527-1531), its use for in vivo selection is limited.
To date, there exist different methods for identifying binding partners in cellular systems. In this context, yeast as a eukaryotic cell is a preferred organism to analyze protein-protein interaction in vivo. One characteristic method using yeast as a preferred organism is the yeast two-hybrid system, which represents a further development of the two-hybrid protein interaction assay (Fields and Song, Nature 349 (1989), 245-246). These cellular methods comprise one binding partner fused to a DNA binding domain, while the respective other binding partner is fused to a strong transcription activation domain, and as a result of the molecular interaction of the partners the generation of a functional transcription factor, leading to a change in phenotype of the cell due to expression of respective genes.
However, since the above-mentioned methods are based on the detection of transcriptional activation, they have several drawbacks such as the generation of false positive responses, since many proteins containing transactivation as well as DNA binding domains can activate the system without a respective interaction. Furthermore, since transcription occurs in the nucleus, the above-mentioned methods are neither appropriate for investigating proteins which are toxic to cells when expressed in the nucleus nor proteins mainly acting in the cytoplasm, because of which those proteins obviously cannot be analyzed.
Hence, there is still a need for assays detecting interactions between proteins and peptides under physiological conditions in living cells.